Novel Blood-Brain Barrier Shuttle Peptides Discovered through the Phage Display Method.

Delivery of therapeutic agents into the brain is a major challenge in central nervous system drug development. The blood-brain barrier (BBB) prevents access of biotherapeutics to their targets in the central nervous system and, therefore, prohibits the effective treatment of many neurological disorders. To find blood-brain barrier shuttle peptides that could target therapeutics to the brain, we applied a phage display technology on a primary endothelial rat cellular model. Two identified peptides from a 12 mer phage library, GLHTSATNLYLH and VAARTGEIYVPW, were selected and their permeability was validated using the in vitro BBB model. The permeability of peptides through the BBB was measured by ultra-performance liquid chromatography-tandem mass spectrometry coupled to a triple-quadrupole mass spectrometer (UHPLC-MS/MS). We showed higher permeability for both peptides compared to N-C reversed-sequence peptides through in vitro BBB: for peptide GLHTSATNLYLH 3.3 × 10-7 cm/s and for peptide VAARTGEIYVPW 1.5 × 10-6 cm/s. The results indicate that the peptides identified by the in vitro phage display technology could serve as transporters for the administration of biopharmaceuticals into the brain. Our results also demonstrated the importance of proper BBB model for the discovery of shuttle peptides through phage display libraries.

Influence of the environment and occupational exposure on the occurrence of Q fever.

Q fever, which is caused by Coxiella bumetii, is a worldwide zoonotic infectious disease and ruminants are the main reservoir for human infections. Humans become infected primarily by inhaling aerosols that are contaminated with C. bumetii. Ingestion (particularly drinking raw milk) and person-to-person transmission are minor routes. Animals shed the bacterium in urine and faeces, and in very high concentrations in birth by-products. The bacterium persists in the environment in a resistant spore-like form which may become airborne and transported long distances by the wind. Q fever is considered primarily an occupational disease of workers in close contact with farm animals or processing their products, however, it may occur also in persons without direct contact. To prevent the introduction and spread of Q fever infection, preventive measures should be implemented including immunisation with currently available vaccines of domestic animals and humans at risk.

Seroprevalence of antibodies to Coxiella burnetii among employees of the Veterinary University in Kosice, eastern Slovakia.

Coxiella burnetii is an obligate intracellular pathogen known to be the causative agent of Q fever, a zoonosis with worldwide occurrence. The organism has been found in many wild and domestic animals. Infected animals shed highly stable bacteria in urine, faeces, milk, and through placental and birth fluids. Humans acquire the infection mainly by inhaling infected aerosols, or by ingesting contaminated raw milk or fresh dairy products; tick transmission has been proven but is probably rare. The aim of the present study was to determine the titres of immunoglobulin IgG against phase I and II of C. burnetii, and to evaluate the risk factors that might be associated with exposure to C. burnetii among employees of the Veterinary University. Venous blood was obtained from 92 employees. IgG antibodies were determined by ELISA method modified in our laboratory using whole cells of the Nine Mile C. burnetii strain. The questionnaire was filled out by every subject to obtain epidemiological and clinical date. Phase I antibodies were detected in 35 subjects, i.e. in 38%, and phase II antibodies in 58 subjects, i.e. in 63%. When using the titre > or = 1:800 as a cut-off level, 2 samples were positive for phase I antibodies (2.1%) and 12 for phase II antibodies (13%). Factors predisposing to infection or exposure to C. burnetii included professional orientation and regular contact with farm animals and pets. Clinical history of some seropositive subjects revealed substantial problems, such as fever of unknown origin, rheumatic disease, disease of heart, liver, respiratory tract (particularly atypical pneumonia), chronic fatigue syndrome and spontaneous abortion in females. Q fever is a profession-related disease and prevention of its spreading within the risk population groups requires observation of basic safety rules.

Serotypes, virulence genes, and PFGE profiles of Escherichia coli isolated from pigs with postweaning diarrhoea in Slovakia.

BACKGROUND: Postweaning diarrhoea (PWD) in pigs is usually the main infectious problem of large-scale farms and is responsible for significant losses worldwide. The disease is caused mainly by enterotoxigenic E. coli (ETEC) and Shiga-toxin producing E. coli (STEC). In this study a total of 101 E. coli isolated from pigs with PWD in Slovakia were characterized using phenotypic and genotypic methods. RESULTS: These 101 isolates belonged to 40 O:H serotypes. However, 57% of the isolates belonged to only six serotypes (O9:H51, O147:H-, O149:H10, O163:H-, ONT:H-, and ONT:H4), including two new serotypes (O163:H- and ONT:H4) not previously found among porcine ETEC and STEC isolated in other countries. Genes for EAST1, STb, STa, LT and Stx2e toxins were identified in 64%, 46%, 26%, 20%, and 5% of isolates, respectively. PCR showed that 35% of isolates carried genes for F18 colonization factor, and further analyzed by restriction endonuclease revealed that all of them were F18ac. Genes for F4 (K88), F6 (P987), F17, F5 (K99), F41, and intimin (eae gene) adhesins were detected in 19 %, 5%, 3%, 0.9%, 0.9%, and 0.9% of the isolates, respectively. The study of genetic diversity, carried out by PFGE of 46 representative ETEC and STEC isolates, revealed 36 distinct restriction profiles clustered in eight groups. Isolates of the same serotype were placed together in the dendrogram, but high degree of polymorphism among certain serotypes was detected. CONCLUSION: Seropathotype O149:H10 LT/STb/EAST1/F4 (14 isolates) was the most commonly detected followed by O163:H- EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18 (five isolates). Interestingly, this study shows that two new serotypes (O163:H- and ONT:H4) have emerged as pig pathogens in Slovakia. Furthermore, our results show that there is a high genetic variation mainly among ETEC of O149:H10 serotype.

Potential association between Coxiella burnetii seroprevalence and selected risk factors among veterinary students in Slovakia.

The present study investigated the prevalence of antibodies to Coxiella burnetii and the possible factors predisposing students of veterinary medicine to C. burnetii infections. IgG antibodies to phase I and phase II C. burnetii antigens were determined by ELISA. Out of 77 students examined, 13 were positive for phase I and 45 were positive for phase II antibodies. The titres determined were in the range of 1 : 100-1 : 3200. Some risk factors may have contributed to the high seroprevalence found in these subjects. For example, there were positive associations with rural life and exposure to the breeding of farm animals, and in addition, work in a dusty environment, such as on fields, gardens, stables and construction sites were also connected to high seroprevalence.

Serological study of Q fever in sheep in the territory of Eastern Slovakia.

Q fever is a zoonosis caused by infection with Coxiella burnetii. Although the reservoir of C. burnetii consists of various species, the most common sources of human infection are farm animals, such as cattle, goats, and sheep. The agent is typically transmitted by the aerosol route, and in more than half of the cases primary infection is symptomless. Clinical outcomes of C. burnetii infection in domestic ruminants consist of abortion and stillbirths in sheep and goats, while in cattle it causes infertility and mastitis. A serological survey for C. burnetii was undertaken on a population of sheep. A total of 269 sheep serum samples were collected and tested for the detection of antibodies against C. burnetii phase I and II antigens using an enzyme-linked immunosorbent assay. The herd investigated was tested twice, i.e. in 2000 and 2009, to detect the changes in seroprevalence. In the first year of investigation, the prevalence of antibodies against C. burnetii phase II antigen was estimated at 37.22% and ten years later at 58.42%. Antibodies against phase I antigen were not detected in any examined serum samples. The difference in seroprevalence after ten years of observation was significant (p = 0.001).

Prevalence of Coxiella burnetii antibodies in wild ruminants in Kavecany zoo, Kosice, eastern Slovakia.

Coxiella burnetii is an obligate intracellular agent that causes Q fever in humans and animals. The most important reservoirs of C. burnetii in nature are small wild rodents, but infection was also demonstrated in other animals, including ruminants. Samples of blood were obtained from 4 mouflons, 60 fallow deer, 9 Cameroun goats, 8 Carpathian goats, and 8 Cameroun sheep living in a zoo. Antibodies to phase I and phase II C. burnetii antigens were determined in sera by ELISA. Antibody titres were detected in the range 1:100-1:200. The serum prevalence of phase II and phase I antibodies to C. burnetii antigens was 25 % and 0% in mouflons, 70 % and 0% in goats, 37.5 % and 12.5 % in sheep and 28.3 % and 5 % in fallow deer, resp. Serologic diagnosis of Q fever in animals can be difficult. Some animals may shed C. burnetii and pose a risk for infection prior to the development of antibodies, and some infected animals never seroconvert. The employed ELISA test is a very sensitive assay for C. burnetii, but it is also a labour intensive method and therefore not routinely available.

Human misfolded truncated tau protein promotes activation of microglia and leukocyte infiltration in the transgenic rat model of tauopathy.

It has been hypothesized that misfolded tau protein could be a mediator of the inflammatory response in human tauopathies. Here we show that neurodegenerative lesions caused by human truncated tau promote inflammatory response manifested by upregulation of immune-molecules (CD11a,b, CD18, CD4, CD45 and CD68) and morphological activation of microglial cells in a rat model of tauopathy. In parallel, the innate immune brain response promotes activation of MHC class II positive blood-borne leukocytes and their influx into the brain parenchyma. These findings have important consequences for the rationale drug development of effective inflammation-based therapeutic strategies for human tauopathies.